The present invention relates to hybrid cell lines capable of continuously producing monoclonal antibodies specific for scrapie-associated fibrils (SAF). The proteins which comprise SAF, also known as PrP's (protease resistant proteins) are observed only in the unconventional slow virus diseases (UNSVD).
UNSVD are progressive degenerative diseases of the central nervous system which are lethal. These diseases naturally affect sheep, goats, mule deer, elk and humans and can be experimentally transmitted to primates and rodents. The human diseases include: Creutzfeldt-Jakob disease (CJD), kuru and Gerstmann Straussler syndrome (GSS), a familiar disease occurring with an autosomal dominant mode of inheritance, while the animal disease in sheep and goats is known as scrapie and the disease of mule deer and elk known as chronic wasting disease. At present, the diagnosis of UNSVD is based on the clinical signs and neuropathology. Conclusive evidence for the diagnosis is the transmission of this disease to animals which may take as long as ten years. SAF are abnormal filamentous structures uniquely and specifically associated with UNSVD. Numerous studies employing tissue from many human neurological diseases and from many experimental and natural animal model systems have shown that SAF are found only in the UNSVD. The SAF are composed of a PrP which in addition to the fibrils is disease specific.
The scrapie-like infectious agents elicit no antibody response in the host, nor does immunosuppression with whole body radiation, cortisone, antileukocytic serum, or cytotoxic drugs alter the incubation period, progress or pattern of disease, or length of illness to death. The scrapie-like infectious agents replicate peripherally, notably in spleen and lymph nodes, but only cause pathology in the central nervous system. In addition, these disease agents do not replicate well in cell culture systems. These disease agents have enormous resistance to the normal physical and chemical virological inactivation systems. This is shown by their resistance to high concentration of formaldehyde or glutaraldehyde and most other antiviral and antiseptic substances and to ultraviolet and ionizing radiation, to ultrasonication and to heat. Numerous organic disinfectants, including detergents and the quaternary ammonium salts, often used for disinfection are inadequate for inactivating these disease agents. They are, however, susceptible to certain proteases and strong denaturing compounds such as phenol, chlorox, potassium permanganate and sodium hydroxide. Thus, much more stringent and harsh procedures must be used to ensure decontamination and disinfection.
The rapid and specific diagnosis of UNSVD will be of tremendous service to the medical community, since human CJD has been transmitted by surgical procedures, organ transplant, electrode implantations, and treatment with biological products such as human growth hormone and dura mater. Patients involved in surgical or transplantation procedures cannot be screened at the present time for the presence of scrapie-like infectious agents in that no test procedure is available. Analogous to blood products relative to AIDS, procedure should be available to screen human biological products for UNSVD agents. This diagnosis is crucial for hospital workers in view of the ever-increasing geriatric population and potential for transmission of the disease to others during surgical procedures. These agents are fatal when transmitted and diagnostic tests are needed which will help in distinguishing the CJD dementias from Alzheimer's disease. Such diagnostic tests, using monoclonal antibodies are provided by this invention.
A key breakthrough in the study of UNSVD was the discovery of an abnormal fibril, scrapie-associated fibrils (SAF), present exculsively in extracts of brains exclusively in these diseases (Merz et al. Acta Neuropathol. 54:63-74, 1981; Merz et al. Nature 306:474-476, 1983; Merz et al. Science 225:437-440, 1984). SAF have been shown to be unique to naturally occurring or experimentally induced UNSVD (Merz et al. Science 225:437-440, 1984). SAF are composed of a modified protease resistant form of a host sialoglycoprotein, PrP (Bolton et al. Science 218:1309-1311, 1982; Merz et al. J. of Virol. 61:42-49, 1987). In experiments designed to isolate infectivity, co-purification of SAF, PrP and infectivity was achieved (Diringer et al. Euro. J. Biochem. 134:555-560, 1983; Prusiner et al. Cell 35:349-358, 1983).
PrP are glycosylated cleavage products of a larger host glycoprotein of 33-35 Kda (Oesch et al. Cell 40:735-746, 1985; Manuelidis et al. Proc. Nat. Acad. Sci. 82:4263-4267, 1985; Rubenstein et al. J. Gen. Virol. 67:671-681, 1986). The detection of this protein in a fibrillar form (SAF) or a non-fibrillar form is diagnostic for UNSVD, (Merz et al. Science 225:437-440, 1984; Brown et al. N. Eng. J. of Med. 314:547-551, 1986). Several isolation procedures have been developed to isolate both SAF and PrP (McKinley et al. Cell 35:57--62, 1983, Helmert and Diringer, Bioscience. Rep. 4:165-170, 1984; Hope et al. EMBO J. 5:2591-2597, 1986). The ability to obtain large quantities of purified PrP and SAF has allowed for the production of polyclonal antibodies to PrP (Bendheim et al. Nature 310:418-421, 1984; Diringer et al. Lancet ii 345, 1984; Kascsak et al. J. of Virol. 59:676-683, 1986; Takahashi et al. Microbiol. Immunol. 30:123-131, 1986). Monoclonal antibodies have been produced to hamster PrP which react exclusively with hamster protein (Barry et al. J. Infect. Dis 154:578-521). These antibodies have been used to specifically identify both SAF and PrP by various immunological means. The western blot procedure has frequently been used to identify PrP in human and animal UNSVD (Bendheim et al. Proc. Nat. Acad. Sci. 82:997-1001, 1985; Bockman et al. N. Eng. J. Med. 312:73-78, 1985; Kascsak et al. J. of Virol. 59:676-683, 1986; Brown et al. N. eng. J. Med. 314:547-551, 1986; Gibbs et al. N. Eng. J. Med. 313:734-738, 1985). SAF have also been identified by immunodecoration (Barry et al. J. of Immun. 135:603-613, 1985; Merz et al. J. of Virol. 61:42-49, 1987). The amyloid present in certain UNSVD also appears to be composed of PrP which differentiates this type of amyloid from amyloids found in other neurological diseases. Antibody to PrP has been used to specifically stain amyloid using immunocytochemistry in UNSVD (DeArmond et al. Cell 41:221-235, 1985; Merz et al. Discussions in Neurosciences 3:458-468, 1986; Roberts et al. N. Eng. J. Med. 315:1231-1232, 1986).